Microorganism for producing polycyclic ether antibiotics

ABSTRACT

An acidic polycyclic ether antibiotic, having structure established by X-ray crystallography, is formed by fermentation of a novel microorganism, Actinomadura sp. ATCC 53764. This novel antibiotic is useful as an anticoccidial in chickens, in the prevention or treatment of swine dysentery, and as a growth promotant in cattle and swine.

This is a division of application Ser. No. 07/655,440, filed on Mar. 27,1992, now U.S. Pat. No. 5,298,524, entitled ACIDIC POLYCYCLIC ETHERANTIBIOTIC HAVING ANTICOCCIDIAL AND GROWTH PROMOTANT ACTIVITY.

BACKGROUND OF THE INVENTION

The present invention concerns a new acidic polycyclic ether antibiotichaving the formula: ##STR1## wherein Me=methyl and Et=ethyl, havingrelative stereochemistry as shown; pharmaceutically acceptable cationicsalts thereof; nutrient feed compositions comprising said antibiotic forpoultry, cattle or swine; its use as an anticoccidial agent in poultry,in the treatment or prevention of swine dysentery, or as a growthpromotant in cattle or swine; a fermentation method for its preparation;and the Actinomadura sp. microorganism which produces said antibiotic insaid fermentation method.

The compound (I) is a new member of the acidic polycyclic ether group ofantibiotics. This family includes such well known agents as monensin(The Merck Index, 10th Ed., Merck and Co.,Inc., Rahway, N.J., 1983,monograph no. 6100), nigericin (loc. cit., monograph no. 6390), narasin(loc. cit., monograph no. 6271), lasalocid (loc. cit., monograph no.5204), and salinomycin (loc. cit., monograph no. 8193). The subject hasbeen reviewed by Westley, "Polyether Antibiotics", Adv. Appl.Microbiol., vol. 22, pp. 177-223 (1977). These compounds are generallyknown as coccidiostats, as feed additive-growth promotants, and/or asagents useful against swine dysentery.

SUMMARY OF THE INVENTION

A culture of Actinomadura sp., ATCC 53764, when fermented under aerobicconditions in aqueous media, produces a new acidic polycyclic etherantibiotic, a compound having the formula (I), as specified above.

The present invention is directed to said compound of the formula (I),including the pharmaceutically-acceptable cationic salts thereof, and toa process for its preparation which comprises fermentation of saidActinomadura sp. ATCC 53764 in an aqueous nutrient medium comprising anassimilable source of carbon and nitrogen until a recoverable amount ofsaid compound of the formula (I) is formed, preferably under submergedaerobic conditions. For use as an anticoccidial agent, in the preventionor treatment of swine dysentery, and/or as a growth promotant, thecompound (I) is not necessarily separated from the fermentation andisolated in substantially pure form, but is alternatively used in crudeform, either in precipitated form admixed with mycelium (recovered byfiltration of the fermentation medium), or in solids obtained by spray-or freeze-drying the entire fermentation medium.

Said pharmaceutically-acceptable cationic salts include, but are notlimited to, those of sodium, potassium, calcium, ammonia,N,N'-dibenzylethylenediamine, N-methylglucamine (meglumine) anddiethanolamine. The preferred cationic salts are those of potassium andsodium.

The present invention is also directed to nutrient feed compositions,one for cattle or swine which comprises the compound of the formula (I)in an amount effective to promote growth and/or improve the feedutilization of said cattle or swine, or to prevent or treat dysentery inswine; and the other for poultry which comprises the compound of theformula (I) in an amount effective to control coccidial infection insaid poultry.

The present invention is further directed to a method for promotinggrowth and/or increasing the efficiency of feed utilization in swine orcattle which comprises administering to said swine or cattle a growthpromoting or feed-utilization efficiency promoting amount of thecompound of the formula (I), particularly in the form of a nutrient feedcomposition; to a method for preventing or treating dysentery in swinewhich comprises administering to said swine a compound of the formula(I) in an amount effective in preventing or treating said dysentery insaid swine; and to a method for controlling coccidial infections inpoultry which comprises administering to said poultry an anticoccidiallyeffective amount of the compound of the formula (I), particularly in theform of a nutrient feed composition.

Finally, the present invention is directed to a biologically pureculture of Actinomadura sp. ATCC 53764, said culture being capable ofproducing the compound of the formula (I) in a recoverable quantity uponfermentation in an aqueous nutrient medium comprising assimilablesources of carbon and nitrogen; including said culture in freeze-driedform.

DETAILED DESCRIPTION OF THE INVENTION

The culture capable of producing the present polycyclic ether antibioticof the formula (I) is designated Actinomadura sp., and was depositedApr. 13, 1988 under the Budapest treaty in The American Type CultureCollection, Rockville, Maryland as the type culture under theiraccession number ATCC 53764. Permanency of the deposit of this cultureat The American Type Culture Collection at Rockville, Maryland and readyaccessibility thereto by the public are afforded throughout theeffective life of the patent in the event the patent is granted. Accessto the culture is available during pendency of the application under 37CFR 1.14 and 35 USC 122. All restrictions on the availability to thepublic of the culture deposited will be irrevocably removed upongranting of the patent.

This novel culture was derived from a soil sample collected in Nigeria,and identified in the culture collection of Pfizer Inc. as N762-11. Itsdescription and classification were provided by Dr. L. H. Huang. Thisculture was found to produce narrow dimensions of the hyphae typical ofthe actinomycetes, an aerial mycelium upon which short spore chains areproduced, and an unfragmented substrate mycelium. The results of thewhole cell analyses further indicate that it belongs to the genusActinomadura.

A slant culture of the microorganism on ATCC 172 media was inoculatedinto ATCC 172 broth and grown for four days at 28° C. on a shaker. Itwas then centrifuged for 20 minutes, washed three times with steriledistilled water, and planted on media commonly used for identificationof members of the Actinomycetales.

The cultures were incubated at 28° C. and the results read at varyingtimes, but most commonly at fourteen days. The colors were described incommon. terminology, but exact colors were determined by comparisonswith color chips from The Color Harmony Manual, fourth edition. Themethods of whole-cell amino acid and sugar analyses are those describedin Becker et al., Appl. Microbiol., vol. 12, pp. 421-423 (1964), and inLechevalier, J. Lab. Clin. Med., Vol. 71, pp. 934-944 (1968),respectively.

The culture was identified as follows:

Yeast Extract-Malt Extract Agar (ISP #2 medium, Difco)--Growth poor tomoderate; white, cream to gray (2 ca, near gray series 3 ih, 3 ml);raised, wrinkled, or appearing as isolated colonies, aerial myceliumwhite to gray (near gray series 3 ih, 3 ml); reverse yellowish gray togray (2 ie, 2 ig, near gray series 3 ih); soluble pigment yellowish gray(2 ie).

Oatmeal Agar (ISP #3 medium, Difco)--Growth moderate, white to cream (2ca), slightly raised, smooth, with white aerial mycelium; reverse cream(2 ca); soluble pigment cream (2 ca).

Inorganic Salts-Starch Agar (ISP #4 medium, Difco)--Growth moderate,white, slightly raised, smooth with white aerial mycelium; reversecolorless to cream (2 ca); no soluble pigment.

Glycerol-Asparagine Agar (ISP #5 medium, Difco)--Growth poor tomoderate, white, slightly raised, smooth to granular, aerial myceliumwhite; reverse colorless; no soluble pigment.

Czapek-Sucrose Agar (Waksman, "The Actinomycetes", v. 2, medium #1, p.328, 1961)--Growth moderate to good, white to cream (2 ca), slightlyraised, smooth, aerial mycelium white; reverse colorless to cream (2ca); no soluble pigment.

Glucose-Asparagine Agar (ibid. , medium #2)--Growth poor, white to cream(2 ca), thin to slightly raised, smooth to granular, aerial myceliumwhite; reverse colorless, cream to pale yellowish (2 ca, 2 ea); nosoluble pigment.

Gordon and Smith's Tyrosine Agar (Gordon and Smith, J. Bacteriol.,69:147-150, 1955)--Growth good, dark pink (4 gc), moderately raised,smooth, no aerial mycelium; reverse pale yellowish brown (3 gc); solublepigment brown (4 ng) .

Calcium Malate Agar (Waksman, Bacteriol. Rev., 21, 1-29, 1957)--Growthpoor, cream (2 ca), thin, smooth to slightly granular; aerial myceliumsparse, white; reverse colorless to cream (2 ca); no soluble pigment.

Casein Agar (Gordon and Smith, ibid.)--No growth. But after four weeksof incubation, growth poor, dark cream (near 2 gc), moderately raised,wrinkled, aerial mycelium white; reverse grayish yellow (2 le); solublepigment grayish (2 ig) .

Bennett's Agar. (Waksman, loc. cit., medium #30, p. 331)--Growth good,gray (near gray series 3 fe, 3 ih) with some white dots, raised,wrinkled, aerial mycelium white to gray (near gray series 3 fe, 3 ih);reverse grayish black (near gray series 3 ih, 3 ml); soluble pigmentyellowish gray (2 ig).

Emerson's Agar (ibid., medium #28, p. 331)--Growth poor to moderate,cream, pale yellowish, yellowish gray to dark gray (2 ca, 2 ea, 2 ia,near gray series 3 ih, 3 ml); raised, wrinkled to granular, aerialmycelium gray (near gray series 3 ih); reverse yellowish to gray (neargray series 2 ih); soluble pigment yellowish gray (2 le, 2 ie).

Nutrient Agar (ibid., medium #14, p. 330)--Growth moderate, paleyellowish to yellowish (2 ea, 2 ga) slightly raised, smooth, no aerialmycelium; reverse same as surface; no soluble pigment.

Gelatin Agar (Gordon and Mihm, J. Bacteriol., 73, 15-27, 1957)--Growthmoderate to good, whitish orange to yellowish orange (3 ea, 3 ia, 4 ea),moderately raised, smooth but wrinkled near edge, no aerial mycelium;reverse yellowish orange (3 ia); no soluble pigment.

Starch Agar (ibid.)--Growth moderate to good, whitish orange toyellowish orange (3 ea, 3 ia), moderately raised, smooth, no aerialmycelium; reverse yellowish orange (3 ia); no soluble pigment.

Potato Carrot Agar (Lechevalier, Lab. Clin. Med., 71, 934-944, 1968, butuse only 30 g. potatoes, 2.5 g. carrots and 20 g. agar)--Growthmoderate, cream (2 ca), thin, smooth; no aerial mycelium; reverse cream(2 ca); no soluble pigment.

Tap Water Agar (2%)--Growth poor to moderate, white, thin, smooth;aerial mycelium white; reverse colorless to cream (2 ca); no solublepigment.

Gauze's Mineral Medium 1 (Gauze et al., Problems in the Classificationof Antagonistic Actinomycetes, English Ed., p. 13, 1957)--Growthmoderate to good, white to cream (2 ca), slightly raised, smooth; aerialmycelium white; reverse cream (2 ca); no soluble pigment.

Gauze's Organic Medium 2 (ibid.)--Growth moderate to good, cream to paleyellowish (2 ca, 2 ea), moderately raised, smooth to wrinkled, aerialmycelium none to sparse, white; reverse same as surface; no solublepigment.

Morphological Properties--The morphological properties were observedafter three weeks of incubation on Gauze's mineral medium 1: aerialmycelium white; spore chains short, flexuous, curved, hooked, or hookedto looped, occasionally coiling into a mass or with open coils of up to1.5 to 2 turns; 3 to 9 spores per spore chain; oval to elliptical,sometimes globose, 0.7-1.0 micron diameter or 1.2-1.8×0.7-1.0 microns;spores warty, as revealed by scanning electron microscopy.

Biochemical Properties--Melanin not produced; hydrogen sulfide notproduced; gelatin liquefied; starch not hydrolyzed; organic nitrate butnot dextrose nitrate reduced to nitrite; poor growth but nodecomposition on either Jensen's or Levine and Schoenlein's cellulosebroth; no coagulation and no peptonization of milk; casein digestionpositive. Carbohydrate utilization: glucose, sucrose, ribose and starchutilized; inositol, galactose, glycerol, melezitose, and trehalosedoubtfully utilized; arabinose, fructose, mannitol, raffinose, rhamnose,xylose, adonitol, cellobiose, dulcitol, erythritol, lactose, maltose,mannose, melibiose, alpha-methyl-D-glucoside, salicin, sorbitol, andsorbose not utilized.

Acid production: acid produced from glucose, arabinose, fructose,mannitol, raffinose, rhamnose, sucrose, xylose, cellobiose, dulcitol,erythritol, lactose, maltose, mannose, melezitose, melibiose, ribose,salicin, sorbitol, sorbose, starch, and trehalose; acid not producedfrom inositol, adonitol, galactose, glycerol, andalpha-methyl-D-glucoside.

The other positive tests included utilization of acetate, citrate,pyruvate, and succinate; and decomposition of hypoxanthine. Thefollowing tests were negative: utilization of benzoate, dextrin,lactate, malate, mucate, oxalate, phenol, and propionate; decompositionof adenine and xanthine; and hydrolysis of hippurate, esculin, and urea;resistance to lysozyme.

    ______________________________________                                        Temperature Relations                                                         21° C.                                                                              28° C.                                                                           37° C.                                                                            45° C.                               ______________________________________                                        Scant to     Good      Good       No                                          Poor Growth  Growth    Growth     Growth                                      ______________________________________                                    

Whole-Cell Analysis--The whole-cell hydrolysates containedmeso-diaminopimelic acid, glucose, galactose, madurose and ribose.

The culture N762-11 is characterized by the white to cream substratemycelium; the short, colorless aerial (sometimes gray) colonies; theshort spore chains which are flexuous, curved, hooked or looped; and thespores with a warty surface. The colors of the substrate mycelium mightrange from colorless, cream, pale yellowish, yellowish, yellowishorange, yellowish gray to gray. Among all of the sugars tested, onlyglucose, sucrose, ribose, and starch were utilized. Hypoxanthine (butnot adenine and xanthine) was decomposed. The culture did not producemelanin and hydrogen sulfide; did not hydrolyze esculin, hippurate,starch, and urea; and was sensitive to lysozyme. The whole-cellhydrolysates contained meso-diaminopimelic acid and the diagnostic sugarmadurose. Thus, the culture N762-11belongs in the genus Actinomadura, asdefined by H. Lechevalier.

The culture N762-11 shows some similarity to the following known speciesof Actinomadura in cultural properties and/or biochemical properties: A.atramentaria, A. livida, and A. macra. It differs from A. atramentariain the warty rather than smooth spores, the absence of dark brownsubstrate mycelium, the utilization of sucrose, the failure to producemelanin and to peptonize milk, and the ability to liquefy gelatin.Compared with A. livida, it differs in the white rather than creamaerial mycelium and the absence of the brown substrate mycelium.

The culture N762-11 differs from Actinomadura macra in the presence ofhooked or looped spore chains, the warty rather than smooth spores, theability to digest casein, the failure to produce hydrogen sulfide, andthe utilization of ribose and starch.

On the basis of the data presented above, the culture N762-11 isconsidered as a member of the genus Actinomadura and designatedActinomadura sp. It has been deposited at the American Type CultureCollection under the accession number ATCC 53764.

The antibiotic compound (I) of the present invention is readily producedby the present Actinomadura sp. by growing at from about 24° to about36° C. under submerged conditions with agitation and aeration on mediaconsisting of carbohydrate sources such as sugars, starches, glycerol;organic nitrogen substances such as soybean meal, casamino acids, yeastextract; growth substances such as grain solubles, fish meal, cottonseed meal; mineral salts containing trace elements such as iron, cobalt,copper, zinc, etc. and calcium carbonate or phosphates as bufferingagents. After growth has been completed, the antibiotic is readilyrecovered by extracting the whole broth with an organic solvent such asn-butanol, methylisobutyl ketone, or chloroform at pH ranges from 4.0 to8.0; by filtering off the mycelium, which contains the precipitatedantibiotic, the filtrate being discarded; or by simply spray-drying orfreeze-drying the whole broth. Alternatively, the mycelium or the wholedried broth is extracted with one of said organic solvents. The purifiedantibiotic compound, if that is desired, is isolated from the organicextract by standard methods of concentration, salt or free acidformation, chromatography, precipitation and/or crystallization, asexemplified below.

In the usual manner of carrying out the fermentation, an inoculum isfirst prepared by scraping vegetative cells, growing on a suitablemedia, from slants or Roux bottles which have been inoculated withActinomadura sp. ATCC 53764. The resulting vegetative cells are in turnused to inoculate shake flasks or inoculum tanks, also containingsuitable growth media. Alternatively, the inoculum tanks are inoculatedfrom the shake flasks. Following a suitable growth period (generally 120to 144 hours in shake flasks and 168 to 196 hours in inoculum tanks), afermenter, also containing suitable growth media, is inoculated underaseptic conditions with vegetative broth from the shake flasks orinoculum tanks. Upon completion of growth (generally about 120-196hours), the antibiotic compound is recovered in crude or pure form, asdesired, by one or another of the methods generally described above, orby specific methods which are exemplified below.

The compound of the formula (I) is tested for in vitro antibacterialactivity by standard methods in which the minimum inhibitoryconcentrations (MIC's) in mcg/ml against one or more microorganisms ismeasured. One such procedure is the one recommended by the InternationalCollaborative Study on Antibiotic Sensitivity Testing (Ericcson andSherris, Acta. Pathologica et Microbiologia Scandinav, Supp. 217,Section B: 64-68 [1971]), and employs brain heart infusion (BHI) agarand an inocula replicating device. Overnight growth tubes are diluted100 fold for use as the standard inoculum (20,000-100,000 cells inapproximately 0,002 ml. are placed on the agar surface; 20 ml. of BHIagar/dish). Twelve 2 fold dilutions of the test compound are employed,with initial concentration of the test drug being 200 mcg/ml. Singlecolonies are disregarded when reading plates after 18 hours at 37° C.The susceptibility (MIC) of the test organism is accepted as the lowestconcentration of compound capable of producing complete inhibition ofgrowth as judged by the naked eye. Like other polycyclic etherantibiotics, the present compound of the formula (I) typically showsGram positive antibacterial activity, as well as activity againstTreponema hyodysenteriae, (the causative agent of swine dysentery) asillustrated in Table I.

                  TABLE I                                                         ______________________________________                                        IN VITRO ANTIBACTERIAL ACTIVITY OF THE                                        COMPOUND OF THE FORMULA (I)                                                   Organism          Strain No.                                                                              MIC, mcg/ml                                       ______________________________________                                        Clostridium perfringens                                                                         10A009    100                                               Actinomyces pyogenes                                                                            14D002    50                                                Treponema hyodysenteriae                                                                        94A001    0.78                                              ______________________________________                                    

Efficacy data for the compound of the formula (I) and its salts againstcoccidial infections in chickens is obtained by the following method.Groups of 3-5 ten-day old pathogen free white leghorn cockerel chicksare fed a mash diet containing the compound (I) or its sodium and/orpotassium salt uniformly dispersed therein. After being on this rationfor 24 hours each chick is inoculated per os with oocysts of theparticular species of Eimeria being tested. Other groups of 3-5 ten-dayold chicks are fed a similar mash diet without compound (I) or itssalts. They are also infected after 24 hours and serve as infectedcontrols. Yet another group of 3-5 ten-day old chicks are fed the samemash diet without antibiotic and are not infected with coccidia. Theseserved as normal controls. The results of treatment are evaluated afterfive days in the case of E. acervulina, and six days for all otherchallenges.

The criteria used to measure anticoccidial activity consists of lesionscores of 0 to 4 for E. tenella after J. E. Lynch, "A New Method of thePrimary Evaluation of Anticoccidial Activity", Am. J. Vet. Res.,22,324-326, 1961; and 0 to 3 for the other species based on modificationof the scoring system devised by J. Johnson and W. H. Reid,"Anticoccidial Drugs. Lesion Scoring Techniques in Battery and Floor PenExperiments in Chicks", Exp. Parasit., 28, 30-36, 1970. Activity ismeasured by dividing the lesion score of each treated group by thelesion score of the infected control. In this test, the compound (I) andits cationic salts exhibit exceptionally high activity against Eimeriatenella, E. acervulina, E. maxima, and E. necatrix infections in poultrywhen incorporated into the mash diet of chickens at levels of about 1.0to 20 ppm. For example, the compound of the formula (I) was some 6 to 24times more effective than salinomycin when compared by this test method.

The present compound of the formula (I) is also generally useful incombination with certain other known anticoccidial agents, such asnicarbazin, 4,4'-dinitrocarbanilide or a naphthalenamine, as defined byHamill et al., U.S. Pat. No. 4,582,822, cited above.

For the prevention or control of coccidiosis in poultry, the compound ofthis invention is orally administered to poultry in a suitable carrier.Conveniently, the medication is simply carried in the drinking water orin the poultry feed, so that a therapeutic dosage of the agent isingested with the daily water or poultry ration. The agent can bedirectly metered into drinking water, preferably in the form of a liquidconcentrate, or added directly to the feed as such, or in the form of apremix or concentrate. A premix or concentrate of therapeutic agent in acarrier is commonly employed for the inclusion of the agent in the feed.The therapeutic agent can be in substantially pure form (e.g., the freeacid, or a pharmaceutically-acceptable salt thereof), in assayed crudeform such as wet or dry mycelium or dried whole broth. Suitable carriersare liquid or solid, as desired, such as water, various meals (forexample, soybean oil meal, linseed oil meal, corncob meal) and mineralmixes such as are commonly employed in poultry feeds. A particularlyeffective carrier is the poultry feed itself; that is, a small portionof poultry feed. The carrier facilitates uniform distribution of theactive materials in the finished feed with which the premix is blended.This is important because only small proportions of the present potentagents are required. It is important that the compound be thoroughlyblended into the premix and, subsequently, the feed. In this respect,the agent may be dispersed or dissolved in a suitable oily vehicle suchas soybean oil, corn oil, cottonseed oil, and the like, or in a volatileorganic solvent and then blended with the carrier. It will beappreciated that the proportions of active material in the concentrateare capable of wide variation since the amount of agent in the finishedfeed may be adjusted by blending the appropriate proportion of premixwith the feed to obtain a desired level of therapeutic agent.

High potency concentrates are blended by the feed manufacturer withproteinaceous carrier such as soybean oil meal and other meals, asdescribed above, to produce concentrated supplements which are suitablefor direct feeding to poultry. In such instances, the poultry arepermitted to consume the usual diet. Alternatively, such concentratedsupplements are added directly to the poultry feed to produce anutritionally balanced, finished feed containing a therapeuticallyeffective level of one or more of the compounds of this invention. Themixtures are thoroughly blended by standard procedures, such as in atwin shell blender, to ensure homogeneity.

For use in poultry, the use levels of the compound described herein willvary under different circumstances. Continuous low-level medication,during the growing period; that is, during the first 5 to 12 weeks forchickens, is an effective prophylatic measure. In the treatment ofestablished infections, higher levels may be necessary to overcome theinfection. The use level of the compound (I) in feed will generally bein the range of about 1.0 to 25 ppm, preferably in the range of about2.5 to 12.5 ppm. When administered in drinking water, the level whichwill be that which will provide the same daily dose of medicationfactored by the weight ratio of the average daily consumption of feed tothe average daily consumption of water.

The activity of the compound of the formula (I) and its salts in thepromotion of growth and/or increasing the efficiency of food utilizationin swine or cattle can be measured directly by feeding test groups ofanimals various levels of the compound (I) or a salt in feed.Alternatively, British Patent Specification No. 1,197,826 details an invitro rumen method for the evaluation of antibiotics in feeds.

For use in the prevention or treatment of swine dysentery, or inpromoting growth and/or increasing the efficiency of feed utilization incattle or swine the compound of the formula (I) or a salt is preferablyadministered as a feed additive. The feeds prepared according to methodsfully analogous to those detailed above for the preparation of poultryfeed, with the same concern for producing feeds in which the therapeuticagent is uniformly dispersed. The use level of the compound (I) incattle or swine feed will generally be in the range of about 0.25 to 25ppm. In ruminants the compound of the formula (I) can also be orallyadministered in the form of a bolus which is retained in therumenoreticular sac, releasing the therapeutic agent at a substantiallyconstant rate over a prolonged period of time, e.g., 4-8 weeks,providing a dose equivalent to that of the above daily dose in feed,i.e.: ##EQU1## Exemplary of such a controlled release bolus is that ofCardinal, U.S. Pat. No. 4,601,893.

The present invention is illustrated by the following examples. However,it should be understood that the invention is not limited to thespecific details of these examples.

EXAMPLE 1 Fermentation of Actinomadura sp. ATCC 53764 Isolation of theAntibiotic of the Formula (I)

The Actinomadrua sp. was initially grown by inoculating solid media onslants or Roux bottles with the ATCC 53764 culture, using ATCC mediumNo. 172, prepared and having composition as follows.

    ______________________________________                                                           Grams/liter                                                ______________________________________                                        Glucose              10                                                       Soluble Starch       20                                                       Yeast Extract         5                                                       Casein Enzymatic Hydrolysate                                                                        1                                                       Calcium Carbonate     1                                                       Distilled Water to 1000 ml;                                                                        20                                                       pH to 7.0 with KOH; Add Agar                                                  ______________________________________                                    

Meanwhile, 300 ml shake flasks were prepared using in each flask 100 mlof one or the other of the following media:

    ______________________________________                                                    Grams/                  Grams/                                    C'          liter    JDYTT          liter                                     ______________________________________                                        Cerelose    10       Cerelose       10                                        Soy Flour   10       Corn Starch    5                                         Corn        5        Corn Steep Liquor                                                                            5                                         Fermentation                                                                  Solids                                                                        Corn Starch 10       Casein Enzymatic                                                                             5                                                              Hydrolysate                                              Sodium Chloride                                                                           5        Cobalt Chloride                                                                              0.002                                     Cobalt Chloride                                                                           0.002    Calcium Carbonate                                                                            3                                         Calcium Carbonate                                                                         1                                                                 ______________________________________                                    

The medium-containing shake flasks were then sterilized at 120° C. and15 p.s.i. for 30 minutes. After cooling, the medium was inoculated witha vegetative cell suspension scraped from the above Actinomadura sp.slant culture. The flasks were shaken at 28° C. on a shaker having adisplacement of 1.5 to 2.5 inches at 150 to 200 cycles per minute (CPM)for five to seven days.

Meanwhile, 5 liter fermentation vessels were prepared containing 3liters of one of the above C' or JDYTT media or the following media:

    ______________________________________                                        UK1-2            Grams/liter                                                  ______________________________________                                        Cerelose         45                                                           Soy Flour        10                                                           Corn Steep Liquor                                                                              10                                                           Cobalt Chloride  0.002                                                        Magnesium Sulfate                                                                              0.10                                                         Calcium Carbonate                                                                              3                                                            Manganese Sulfate                                                                              0.10                                                         Ferric Sulfate   0.10                                                         ______________________________________                                    

An antifoaming agent (polypropyleneglycol, P2000, containing 10%ethylene oxide by weight, 1 ml) was added, and the vessels were sealedand sterilized at 120° C. and 15 p.s.i. for 45 minutes. The vessels werethen inoculated with one shake flask (ca 3% inoculum), and fermented for120 to 168 hours at 30° C., stirring at 1700 revolutions per minute(RPM) with an air rate of one volume of air per volume of liquid perminute.

When the fermentation was completed (based on an antibiotic disc assayversus B. subtilis ATCC 6633) the fermenters were stopped and filteredat the natural pH with the aid of a diatomaceous earth. The filter cakewas slurried in methanol, concentrated in vacuo, diluted with 2-3volumes of water then extracted 2X with 1/3 to 1/2 volume of eithermethylisobutyl ketone or n-butanol. The solvent layer was separated fromthe aqueous phase by aspiration or centrifugation, sparkled andconcentrated in vacuo to yield the antibiotic of the formula (I) incrude form as a viscous oil.

The bioactivity of the broth and subsequent recovery streams can befollowed by using a sensitive strain of Bacillus subtilis ATCC 6633 orStaphylococcus aureus ATCC 6538. The components in the broth andrecovery streams can be visualized by thin layer chromatography (tlc)using Analtech silica gel GF plates employing ethyl acetate as eluant.The developed plates are sprayed with vanillin reagent (3 g vanillin in75 ml ethanol and 25 ml 85% phosphoric acid) and heated to 80° C. Theantibiotic product of the formula (I) appears as an orange spot. Thedeveloped tlc plate can also be overlayed with agar seeded with eitherS. aureus or B. subtilis to which 2,3,5-triphenyl-2H-tetrazoliumchloride monohydrate has been added and incubated at 37° C. for 16 hoursto visualize the antibiotic (white spots against a pink background).

Scale-up in large fermentation vessels was carried out by preparingshake flasks containing 0.7 liters of C' or JDYTT medium. The shakeflask inoculum was fermented for 5 to 7 days at 28° C., and used toinoculate a 200 or a 4000 liter fermentation vessel containing 100 or4000 liters of UK1-2 medium, respectively. Approximately one liter ofinoculum was used in each tank. The fermentations, after proceeding for7 to 10 days, were harvested.

The whole broth of the smaller fermentation run was extracted with 33liters of methylisobutyl ketone at natural pH. The organic extract wasseparated on an alpha-DeLaval separator and concentrated under vacuum toyield the crude antibiotic of the formula I as an oil.

Work-up of the large tank fermentation was carried out by extracting theapproximately 4000 liters of whole broth with 1400 liters ofmethylisobutyl ketone. The organic extract was separated andconcentrated under vacuum, initially in a vacuum pan and finally on arotary evaporator to yield 12 liters of crude product as a syrup. Thesyrup was extracted 2×25 liters of methanol. The extracts were combinedand restripped to a second oil which was chromatographed on SephadexLH20, monitored by tlc as described above. Product containing cuts werecombined and chromatographed on silica gel, eluted sequentially withhexane, toluene, CHCl₃, ethyl acetate and acetone, again monitoring bytlc. The desired product was contained in the CHCl₃ and ethyl acetatecuts. The latter were combined, concentrated to 85 g of residue andrechromatographed in silica gel using 1:1 ethyl acetate: hexane aseluant. Product fractions were combined, treated with activated carbon,filtered, shaken with pH 9.0 dibasic sodium phosphate buffer from thesodium salt, dried over Na₂ SO₄, stripped and the 2.03 g of antibioticof the formula (I) recovered as the sodium salt.

In a second 4000 liter fermentation the initial crude syrup wasextracted 2×25 liters of 1:3 methanol: hexane, the extracts combined andstripped to an oil. The latter was chromatographed on 3 Kg of silicagel, initially eluting with hexane, then ethyl acetate and finally 9:1ethyl acetate: acetone. Product containing fractions, identified by tlc,were combined, stripped, taken up in ethyl acetate, treated withactivated carbon, filtered, and extracted with dilute phosphoric acidand then with pH 9.0 phosphate buffer. The organic phase was dried (Na₂SO₄), stripped, and the residue crystallized by trituration with heptaneand dried under high vacuum to yield 2.2 g of the antibiotic of theformula (I) in the form of its sodium salt; mp 265°-267° C.; [alpha]_(D)²⁵ =+31.2° (c=1, CHCl₃).

Anal. Calcd. for C₅₀ H₈₅ O₁₆ Na.2H₂ O: C, 59.98; H, 8.95 Found: C,59.61; H, 8.56.

EXAMPLE 2 Compound (I) in the Free Acid Form

The free acid form of the antibiotic of the formula (I) was prepared byvigorously shaking a chloroform solution of the sodium salt with anequal volume of hydrochloric acid at pH 2 in a separatory funnel. Thephases were separated, and the chloroform layer was washed with waterand then evaporated under vacuum to give the free acid: mp 115°-117° C.;[alpha]_(D) ²⁵ =+21.6° (c=1, CHCl₃)

Anal. Calcd for C₅₀ H₈₆ O₁₆ : C, 63.67; H, 9.19 Found: C, 63.52; H,9.36.

C-13 nmr (chemical shift (ppm) in CDCl₃ with number of hydrogens inparentheses): 177.0 (0) , 107.7 (0), 101.5 (1), 97.6 (1), 96.9 (0), 89.2(0), 85.5 (0), 84.9 (1), 84.4 (1), 82.4 (1), 80.5 (1), 80.4 (1), 76.7(1), 75.1 (1), 74.8 (1), 74.6 (1), 73.9 (1), 71.0 (1), 68.1 (2), 67.2(1), 56.9 (3), 56.8 (3), 41.3 (1), 38.5 (2), 37.5 (1), 36.6 (2), 36.0(1), 34.8 (1), 34.6 (1), 34.0 (2), 33.6 (2), 32.8 (1), 32.8 (2), 30.8(2), 30.8 (2), 27.8 (3), 27.6 (2), 27.4 (2), 27.3 (2), 26.6 (2), 24.4(2), 18.3 (3), 18.3 (3), 17.4 (3), 16.2 (3), 16.0 (3), 15.5 (3), 10.7(3), 10.6 (3), and 10.1 (3).

The free acid was recrystallized by slow evaporation from diethyl etherand the X-ray structure was determined on the resulting crystals by Dr.J. Bordner.

EXAMPLE 3 The Sodium Salt of the Compound (I)

The free acid of the preceding Example (130 mg) was dissolved in 100 mlof chloroform. A solution of sodium carbonate (0.3 g) in water (100 ml)was added and the resulting mixture was then placed in a separatoryfunnel and vigorously shaken for several minutes. The chloroform layerwas separated, washed with water, dried over sodium sulfate, filtered,and evaporated to afford 136 mg of the sodium salt; having mp and otherproperties identical to the sodium salt described above.

EXAMPLE 4 The Potassium Salt of the Compound (I)

To prepare the potassium salt of the antibiotic compound of the formula(I), the free acid (120 mg) was dissolved in 100 ml of chloroform. K₂CO₃ (0.5 g) in 100 ml of H₂ O was added and the resulting mixturestirred for several minutes, then placed in a separatory funnel andvigorously shaken for several minutes. The organic phase was separated,washed with water and evaporated under vacuum to afford title product;mp 130°-135° C., [alpha]_(D) ²⁵ =+22.8° (c=1, chloroform).

Anal. Calcd. for C₅₀ H₈₅ O₁₆ K: C, 61.14; H, 8.66. Found: C, 61.89; H,8.98.

We claim:
 1. A biologically pure culture of Actinomadura sp. ATCC 53764which produces the compound of the formula ##STR2## wherein Me=methyland Et=ethyl in a recoverable quantity upon fermentation in an aqueousnutrient medium comprising assimilable sources of carbon and nitrogen.2. The culture of claim 1 in freeze-dried form.
 3. A biologically pureculture of Actinomadura sp. 53764.